Pathological hypertrophy is a common predecessor to heart failure (HF). The heart also grows in response to exercise but this growth, termed physiological hypertrophy, does not generally lead to adverse consequences and can even protect the heart against pathological stress. There is a fundamental gap in our understanding of why cardiac hypertrophy can have such divergent outcomes. Our over-arching hypothesis is that there are distinct forms of hypertrophy, which may appear superficially similar but have dramatically different likelihoods of progressing to HF. Our long-term goal is to understand the pathways responsible for these differences and learn whether they can be exploited therapeutically. The objective of the current application is to understand the role of the microRNA (miRNA), miR-222, in pathological hypertrophy and HF. Prior work from the applicant's laboratory identified 16 cardiac miRNAs that were concordantly regulated in two distinct exercise models. Of these, miR-222, which is also increased in serum of HF patients after exercise, was necessary for exercise-induced physiological cardiac growth. While miR-222 was not sufficient to induce cardiac hypertrophy at baseline, it was sufficient to protect against adverse remodeling after ischemic injury. The role of miR-222 in pathological hypertrophy and HF remains unexplored. Based on preliminary data presented in this application, we hypothesize that miR-222 ? despite being involved in physiological hypertrophy ? paradoxically protects against pathological hypertrophy and the progression to HF. Moreover, we hypothesize that miR-222 acts as a nodal modulator of physiological versus pathological genetic programs at least in part through effects on two transcription factors: HMBOX1 and NFATc3. These central hypotheses will be tested in three integrated Specific Aims. In Aim 1, we will use specific and effective gain- and loss-of-function models to directly assess the role of miR-222 in pathological hypertrophy and HF. In Aim 2, a combination of expression profiling and bioinformatic analyses will be used to identify downstream targets of miR-222 and delineate the mechanisms responsible for its effects in pathological hypertrophy and HF. In Aim 3, a novel technology termed CombiGEM (Combinatorial Genetics En Masse), recently developed by our collaborator, Dr. Tim Lu of the MIT Synthetic Biology group, will be used to investigate the additive or synergistic effects of miRNAs altered in exercised hearts. In vivo studies will be supported by in vitro investigation of primary cardiomyocytes to elucidate the underlying mechanisms. Our approach combines innovative hypotheses, technologies, and unique animal models with the complementary expertise of an outstanding team of collaborating investigators. The proposed research is significant, because it is expected to advance our understanding of cardiac hypertrophy and HF as well as pathways with the potential to mitigate these clinically important conditions.